Pentachlorophenol (PCP) is one of the most widely used biocides, primarily for wood preservation. PCP is now considered to be ubiquitously present in our environment and even found in people who are not occupationally exposed to it. PCP has been listed as a priority pollutant by the U. S. Environmental Protection Agency (EPA), and classified as a Group 2B environmental carcinogen by the International Association of Research on Cancer (IARC). The genotoxicity of PCP has been attributed to its two major metabolites: tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone. Recently, the investigators found that hydroxyl radicals (-OH) are produced by these PCP metabolites through a novel, metal-independent mechanism (the so-called organic Fenton reaction); and that dietary antioxidants markedly inhibit -OH production from PCP metabolites. These preliminary in vitro data suggest that dietary antioxidants may play an important role in modulating the genotoxicity of PCP metabolites. Therefore, in this proposal, the researchers plan to extend their studies from a purely chemical system to a cell culture system, using human fibroblasts. Specifically, the following questions will be addressed: (1) Can this novel mechanism of metal-independent -OH production be detected in cells? The production of -OH will be measured by secondary radical ESR spin-trapping techniques; and the role of metals will be studied by using specific intra-and extracellular iron and copper chelating agents. (2) Can dietary antioxidants, viz., lipoic acid and ascorbic acid, protect against cytotoxicity and genotoxicity induced by PCP metabolites? Cytotoxic and genotoxic effects of PCP metabolites will be evaluated, respectively, by the MTT assay and the comet assay. (3) What are the molecular mechanisms of the modulatory effects of these dietary antioxidants? The interaction between PCP metabolites and antioxidants will be studied by various spectral methods (UV-visible, ESR) and by high-pressure liquid chromatography with photodiode array or electrochemical detection. This project should result in a better understanding of the molecular mechanisms underlying the cytotoxic and genotoxic effects of PCP metabolites and related environmental agents, and may lead to novel strategies for the prevention and treatment of these toxic effects by dietary antioxidants.